Bactericidal methods and compositions

ABSTRACT

Methods of the present invention comprise photoinactivation of catalase in combination with low-concentration peroxide solutions and/or ROS generating agents to provide antibacterial effects.

RELATED APPLICATIONS/PATENTS

This application is a continuation of U.S. patent application Ser. No.17/005,807 filed on Aug. 28, 2020 which is a continuation of and claimspriority under 35 U.S.C. § 120 of co-pending International PatentApplication No. PCT/US2020/016125 filed on Jan. 31, 2020, which claimsthe benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationSer. No. 62/799,328 filed on Jan. 31, 2019, the contents of each ofwhich are incorporated herein by reference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by a National Institutes of Health Grant No.R11AI132638. The government may have certain rights to the invention.

BACKGROUND OF THE INVENTION

Antibiotic resistance kills an estimated 700,000 people each yearworldwide, and studies predict that this number could rise to 10 millionby 2050, if efforts are not made to curtail resistance (Willyard, C. J.N. N. The drug-resistant bacteria that pose the greatest health threats.543, 15 (2017)). Yet, the pace of resistance acquisition from mutationin pathogens is faster than clinical introduction of new antibiotics.There is an urgent need to develop unconventional ways to combat theresistance.

SUMMARY OF THE INVENTION

The lethal effect of certain antibiotics occurs through the generationof Reactive Oxygen Species (ROS). Catalase, the ubiquitous key defenseenzyme existing in most of the aerobic pathogens, is utilized toscavenge hydrogen peroxide, thus preventing downstream oxidative damage.It has now been shown that catalase can be optimally photoinactivated byblue light having a wavelength of about 400 nm to about 430 nm, andspecifically, a wavelength of about 410 nm. Photoinactivation ofcatalase renders broad-SPECTRUM catalase-positive microbial pathogenshighly susceptible to ROS-generating antimicrobials and/or immune cellattack. It has now been further determined that the antimicrobial effectof photoinactivation is significantly and unexpectedly increased uponadministration of a low-concentration of H₂O₂ and/or a ROS-generatingagent.

In one aspect, the invention provides a method of treating a tissue of asubject infected with a catalase-positive microbe, said methodcomprising the steps of: applying light to the tissue of the subjectinfected with the catalase-positive microbe at a wavelength of about 400nm to about 430 nm, wherein the catalase is inactivated, and contactingthe tissue with a composition comprising a diluted peroxide solution,thereby treating the tissue of the subject infected with thecatalase-positive microbe.

In one embodiment, the wavelength is about 410 nm.

In another embodiment, the dose of the light is about 5 J/cm² to about200 J/cm².

In yet another embodiment, the dose of the light is about 15 J/cm².

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a pulsed nanosecondlaser.

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a continuous wave LED.

In yet another embodiment, the diluted peroxide solution is a hydrogenperoxide solution.

In yet another embodiment, the hydrogen peroxide solution is betweenabout 0.03% and about 0.3% hydrogen peroxide.

In yet another embodiment, the method further comprises administering aROS generating agent to the infected tissue of the subject.

In yet another embodiment, the ROS generating agent is tobramycin,silver cation, iodine tincture, a gold nanoparticle, methylene blue, aβ-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, amembrane-targeting polyene antifungal or a cell-wall targetingantifungal.

In yet another embodiment, the tissue is skin, scalp or nails.

In yet another embodiment, the catalase-positive microbe is eradicated.

In another aspect, the invention provides a method of disinfecting aninanimate surface contaminated with a catalase-positive microbe, saidmethod comprising the steps of: applying light to the inanimate surfaceat a wavelength of about 400 nm to about 430 nm, wherein the catalase isinactivated, and contacting the inanimate surface with a compositioncomprising a diluted peroxide solution, thereby disinfecting theinanimate surface.

In one embodiment, the wavelength is about 410 nm.

In another embodiment, the dose of the light is about 5 J/cm² to about200 J/cm².

In yet another embodiment, the dose of the light is about 15 J/cm².

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a pulsed nanosecondlaser.

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a continuous wave LED.

In yet another embodiment, the diluted peroxide solution is a hydrogenperoxide solution.

In yet another embodiment, the hydrogen peroxide solution is betweenabout 0.03% and about 0.3% hydrogen peroxide.

In yet another embodiment, the method further comprises administering aROS generating agent to the infected tissue of the subject.

In yet another embodiment, the ROS generating agent is tobramycin,silver cation, iodine tincture, a gold nanoparticle, methylene blue, aβ-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, amembrane-targeting polyene antifungal or a cell-wall targetingantifungal.

In yet another embodiment, the inanimate surface is a materialcomprising metal, plastic, fabric, rubber, stone, composite surfaces orwood.

In yet another embodiment, the catalase-positive microbe is eradicated.

In yet another aspect, the invention provides a method of treating atissue of a subject infected with a catalase-positive microbe, saidmethod comprising the steps of: applying light from a pulsed nanosecondlaser to the tissue of the subject infected with the catalase-positivemicrobe at a wavelength of about 400 nm to about 460 nm, wherein thecatalase is inactivated, and contacting the tissue with a compositioncomprising a ROS generating agent, thereby treating the tissue of thesubject infected with the catalase-positive microbe.

In one embodiment, the wavelength is about 410 nm.

In another embodiment, the dose of the light is about 5 J/cm² to about200 J/cm².

In yet another embodiment, the dose of the light is about 15 J/cm².

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe.

In yet another embodiment, the diluted peroxide solution is a hydrogenperoxide solution.

In yet another embodiment, the hydrogen peroxide solution is betweenabout 0.03% and about 0.3% hydrogen peroxide.

In yet another embodiment, the ROS generating agent is tobramycin,silver cation, iodine tincture, a gold nanoparticle, methylene blue, aβ-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, amembrane-targeting polyene antifungal or a cell-wall targetingantifungal.

In yet another embodiment, the tissue is skin, scalp or nails.

In yet another embodiment, the catalase-positive microbe is eradicated.

In yet another aspect, the invention provides a method of producing asynergistic antimicrobial effect in a tissue of a subject infected witha catalase-positive microbe, said method comprising the steps of:applying light to the tissue of the subject infected with thecatalase-positive microbe at a wavelength of about 400 nm to about 460nm, wherein the catalase is inactivated, and contacting the tissue witha composition comprising a diluted peroxide solution, thereby producingthe synergistic antimicrobial effect in the tissue of the subjectinfected with the catalase-positive microbe.

In one embodiment, the wavelength is about 410 nm.

In another embodiment, the dose of the light is about 5 J/cm² to about200 J/cm².

In yet another embodiment, the dose of the light is about 15 J/cm².

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a pulsed nanosecondlaser.

In yet another embodiment, the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a continuous wave LED.

In yet another embodiment, the diluted peroxide solution is a hydrogenperoxide solution.

In yet another embodiment, the hydrogen peroxide solution is betweenabout 0.03% and about 0.3% hydrogen peroxide.

In yet another embodiment, the method further comprises administering aROS generating agent to the infected tissue of the subject.

In yet another embodiment, the ROS generating agent is tobramycin,silver cation, iodine tincture, a gold nanoparticle, methylene blue, aβ-lactam antibiotic, an aminoglycoside, a fluoroquinolone, an azole, amembrane-targeting polyene antifungal or a cell-wall targetingantifungal.

In yet another embodiment, the tissue is skin, scalp or nails.

In yet another embodiment, the catalase-positive microbe is eradicated.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, incorporatedherein by reference.

FIG. 1A-1B depicts the effect of ns-410 nm exposure on pure catalasesolution. (FIG. 1A). Absorption spectra of pure catalase solution underns-410 nm exposure. Catalase solution: 3 mg/ml, filtered with a 0.2 μmfilter. (FIG. 1B). Percent of remaining active catalase after differenttreatment schemes (different wavelengths under the same dosage).Quantification of catalase was obtained by an Amplex Red Catalase kit.Data: Meanfstandard deviation (N=3).

FIG. 2A-2B depicts the effect of ns-410 nm exposure on active catalasepercentages from MRSA USA300 and P. aeruginosa. (FIG. 2A-2B). Percent ofactive catalase remained inside MRSA USA300 (FIG. 2A) and P. aeruginosa(FIG. 2B) after different treatment schemes (different wavelengths underthe same dosage). Quantification of catalase was obtained by an AmplexRed Catalase kit. Data: Mean±standard deviation (N=3).

FIG. 3 depicts Resonance Raman spectra of bovine liver catalase powderwith and without 410 nm exposure. 410 nm dose: 250 mW/cm2. Ramanspectrum acquisition time: 25 s. 532 nm excitation. Data: Mean±SD fromfive spectra.

FIG. 4A-4C depicts the comparison between ns-410 nm and CW-410 nmexposure on the catalase photoinactivation effect from pure catalasesolution (FIG. 4A), catalase from MRSA USA300 (FIG. 4B), and catalasefrom P. aeruginosa (FIG. 4C). Quantification of catalase was obtained byan Amplex Red Catalase kit. Data: Meanfstandard deviation (N=3). Studentunpaired t-test, ***: p<0.001: **: p<0.01.

FIG. 5A-5C depicts CFU ml−1 of stationary-phase MRSA USA300methicillin-resistant Staphylococcus aureus (FIG. 5A), Pseudomonasaeruginosa (FIG. 5B), and Salmonella enterica (FIG. 5C) under thetreatment of 22 mM H₂O₂ with/without the combination with various lightexposure. Data: Meanfstandard deviation (N=3). Student unpaired t-test,***: p<0.001; **: p<0.01. 250 CFUs: limit of detection. FIG. 5A-5Bfurther depicts the synergistic effect between photoinactivation ofcatalase and low-concentration hydrogen peroxide to eliminatestationary-phase MRSA USA300 and stationary-phase Pseudomonasaeruginosa. FIG. 5A-5B: CFU ml-1 of stationary-phase MRSA and P.aeruginosa under different treatment schemes, respectively. N=3. Data:Mean±SD. ***: significant difference. p<0.001. 250 CFUs: detection oflimit.

FIG. 6 depicts the killing efficacy comparison between CW-410 nm andns-410 nm combined with H₂O₂ in both stationary-phase MRSA USA300 andPseudomonas aeruginosa. Left and right: CFU ml−1 of stationary-phaseMRSA and P. aeruginosa under different treatment schemes, respectively.N=3. Data: Mean±SD. ***: significant difference. p<0.001. 250 CFUs:detection of limit.

FIG. 7 depicts CFU ml−1 of E. coli BW25113 under different treatmentschemes. Tobramycin: 2 μg/ml, 4-hour incubation. ***: p<0.001, studentunpaired t-test.

FIG. 8 depicts CFU ml−1 of Enterococcus faecalis NR-31970 underdifferent treatment schemes. Tobramycin: 2 μg/ml, 4-hour incubation.

FIG. 9A-9H depicts confocal laser scanning microscopy of intracellularMRSA. (FIG. 9A-9C). Fluorescence images of intracellular live MRSA (FIG.9A), and dead MRSA (FIG. 9B), along with the transmission images (FIG.9C) after MRSA infecting RAW 264.7 macrophage cells for 1 hour. (FIG.9D-9F). Fluorescence images of intracellular live MRSA (FIG. 9D), anddead MRSA (FIG. 9E), along with the transmission images (FIG. 9F) afterns-410 exposed MRSA infecting RAW 264.7 macrophage cells for 1 hour.(FIG. 9G-9H). Quantitative analysis of live/dead MRSA from the above twogroups. Scalar bar=10 μm.

FIG. 10 depicts active catalase percent of various fungal strains withor without 410 nm light exposure. Dose: 410 nm, 150 mW/cm2, 5 min.Fungal concentration: 106 cells/ml. C. albicans CASC5314: wild-typeCandida albicans.

FIG. 11 depicts CFU results of C. albicans CASC5314 after differenttreatment schemes. (Left) Time-killing assay of CASC5314 after varioustreatment schemes. (Right) Spread plates of CASC5314 after 1-hourincubation at different treatment schemes.

FIG. 12 depicts the synergistic effect between photoinactivation ofcatalase under various wavelengths and low-concentration hydrogenperoxide to eliminate stationary-phase CASC5314. CFU ml−1 of CASC5314after treatments under the combination between H₂O₂ and variouswavelengths. Dosage: 40 mW/cm², 24 J/cm². H₂O₂: 44 mM, 1.5-hourincubation. Data. Mean±SEM (N=3). ##: detection limit.

FIG. 13A-13D depicts fluorescence signals of PrestoBlue from CASC5314under various treatment schemes. (FIG. 13A, 13C). H₂O₂-alone treatedstationary-phase CASC5314 and log-phase CASC5314, respectively. (FIG.13B, 13D). 410 nm plus H₂O₂ treated stationary-phase CASC5314 andlog-phase CASC5314, respectively.

FIG. 14 depicts Fluorescence signals of PrestoBlue of three different C.auris strains under different treatment schemes: Amp B alone-treatedgroups and 410 nm plus amp B-treated groups.

FIG. 15 depicts confocal laser scanning imaging of live/dead C. albicansafter infecting RAW264.7 macrophage cells.

FIG. 16 depicts photoinactivation of catalase in combination with silvercation kills MRSA. Shown in the images are spread agar plates of MRSAUSA300 under different treatment schemes.

FIG. 17A-17B depicts the comparison between CW-410 and ns-410 toinactivate catalase and eliminate E. coli BW25113 by synergizing withsilver cation. (FIG. 17A-17B). CFU ml⁻¹ of E. coli BW251 13 afterdifferent treatment schemes: 30 min for (FIG. 17A) and 60 min for (FIG.17B). Dose: 22 J/cm². Silver cation: 0.5 μM. Data: Mean f SEM (N=3).***: p<0.001, significant difference. Student unpaired t-test.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control.

As used herein, the phrase “treating an infected tissue” refers tocuring, alleviating or partially arresting the clinical manifestationsof the infection or its complications. Treating an infected tissueachieves a medically desirable result. In some cases, this is a completeeradication of infection. In other cases, it is an improvement in thesymptoms of the infection.

A “ROS-generating agent” is any biological or chemical agent thatproduces Reactive Oxygen Species (ROS). ROS-generating agents as definedherein, exclude exogenous photosensitizer agents that have beenlight-activated. A “photosensitizer” is a chemical compound, or abiological precursor thereof, that produces a phototoxic or otherbiological effect on biomolecules upon photoactivation.

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle and higherprimates.

A “microbe” is a multi-cellular or single-celled microorganism,including bacteria, protozoa, and some fungi and algae. The termmicrobe, as used herein, includes pathogenic microorganisms such asbacterium, protozoan, or fungus.

The term “inanimate surface” refers any non-living surface.

The term “disinfecting” refers to destroying or eliminating pathogenicmicroorganisms that cause infections.

Unless specifically stated or clear from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” isunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. A range of 1 to 50 is understood to include anynumber, combination of numbers, or sub-range from the group consistingof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well asfractions thereof unless the context clearly dictates otherwise). Forexample, the wavelengths from about 400 nm to about 460 nm include thewavelengths 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411,412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439,440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,554, 455, 456, 457, 458, 459 and 460 nms. The light from about 5J/cm²-to about 200 J/cm² includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 J/cm².

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention

Hydrogen peroxide (H₂O₂) is continuously produced inside microbes fromautoxidation of the redox enzyme, and it diffuses quickly into theintracellular environment, causing an acutely detrimental effect (e.g.lipid peroxidation, DNA and protein damage) as a result of the Fentonreaction:

Fe2++H₂O₂→Fe3++. OH+OH−

Fe3++H₂O₂→Fe2++. OOH+H+

Photoinactivation of catalase creates potent antimicrobial effects dueto a lethal accumulation of ROS. Photoinactivation of catalase furtherassists immune cells to eliminate intracellular pathogens. Neutrophilsand macrophages are highly motile phagocytic cells that serve as thefirst line of defense of the innate immune system (Segal, A. W., AnnuRev Immunol 23, 197-223, doi:10.1 146/annurev.immunol.23.021704.115653(2005)). These cells play an essential role in providing resistance tobacterial and fungal infections through releasing ROS burst (e.g.,superoxide, hydroxyl radicals, and singlet oxygen (Hampton, M. B., Blood92, 3007-3017 (1998)). However, pathogens possess an array of elaboratestrategies to invade and survive inside neutrophils or macrophages, thusacting as the ‘Trojan horses’ responsible for further dissemination andrecurrent infections (Lehar, S. M. et al. Nature 527, 323-328 (2015)).Catalase, which is encoded by gene, katA, confers indispensableresistance for antimicrobial agents or reactive oxygen species releasedby immune cells (Flannagan, R., Pathogens 4, 826-868 (2015)).Photoinactivation of catalase assists macrophage and neutrophils toreduce the intracellular and extracellular bacterial burden.

In conducting the methods of the present invention, photoinactivation ofcatalase is preferably conducted with light having a wavelength of about400 nm to about 430 nm, in combination with administration of alow-concentration peroxide solution and/or an ROS generating agent.Methods of the invention exclude the use of exogenous photosensitizingagents that have been activated by light.

Peroxide solutions include, but are not limited to solutions containinghydrogen peroxides, metal peroxides, and organic peroxides. Hydrogenperoxides include, but are not limited to, peroxy acids,peroxymonosulfuric acid, peracetic acid, peroxydisulfuric acid,peroxynitric acid, peroxynitrous acid, perchloric acid, andphthalimidoperoxycaproic acid. Metal peroxides include but are notlimited to ammonium periodate, barium peroxide, sodium peroxide, sodiumperborate, sodium persulfate, lithium peroxide, magnesium peroxide,magnesium perchlorate and zinc peroxide. Organic peroxides include butare not limited to acetone peroxide, acetozone, alkenyl peroxidearachidonic acid 5-hydroperoxide, artelinic acid, artemether,artemisinin, artemotil, arterolane, artesunate, ascaridole, benzoylperoxide, bis(trimethylsilyl) peroxide, tert-butyl hydroperoxidetert-butyl peroxybenzoate, CSPD([3-(1-chloro-3′-methoxyspiro[adamantane-4,4′-dioxetane]-3′-yl)phenyl]dihydrogen phosphate), cumene hydroperoxide, di-tert-butyl peroxide,diacetyl peroxide, diethyl ether peroxide, dihydroartemisinin,dimethyldioxirane, 1,2-dioxane, 1,2-dioxetane, 1,2-dioxetanedione,dioxirane, dipropyl peroxydicarbonate, ergosterol peroxide,hexamethylene triperoxide diamine, methyl ethyl ketone peroxide,nardosinone, paramenthane hydroperoxide, perfosfamide, peroxyacetylnitrate, peroxyacyl nitrates, prostaglandin h2, 1,2,4-trioxane, andverruculogen. Other peroxides include, potassium peroxydisulfate,bis(trimethylsilyl) peroxide (Me₃SiOOSiMe₃), phosphorus oxides, ammoniumperoxide, copper(II) peroxide, sodium peroxide, cobalt(II) peroxide,mercury(I) peroxide, iron(II) peroxide potassium peroxide, copper(I)peroxide, rubidium peroxide, cesium peroxide, iron(III) peroxide,beryllium peroxide, magnesium peroxide, nickel(H) peroxide, cadmiumperoxide, barium peroxide, benzoyl peroxide, calcium peroxide, diacetylperoxide, cesium superoxide, lead(IV) peroxide, lithium peroxide,gallium(I1) peroxide, chromium(III) peroxide, mercury(II) peroxide,gold(I) peroxide, strontium peroxide, zinc peroxide, potassiumsuperoxide, and chromium(VI) peroxide.

In other specific embodiments, the diluted peroxide solution is ahydrogen peroxide solution formulated with between about 0.030% andabout 0.3% hydrogen peroxide (which converts to about 8.8 mM to about 88mM hydrogen peroxide).

Photoinactivation of catalase and administration of the peroxidesolution can also be provided in combination with ROS generating agentsincluding antibiotics, such as tobramycin. Other ROS generating agentsinclude, but are not limited to, silver cation, iodine tincture, goldnanoparticles, methylene blue (non-photoactivated), β-lactamantibiotics, aminoglycosides, fluoroquinolones, antifungal azoles,membrane-targeting polyene antifungals, such as amphotericin B, andcell-wall targeting antifungals, such as caspofungin.

Typically following photoinactivation, the peroxide solution can beadministered to the site of the infection for a duration of about 10 toabout 30 minutes. In alternate embodiments, the peroxide solution, theROS generating agent and/or the photoinactivating light can beadministered concomitantly or sequentially to the site of infection. Forexample, in specific embodiments, the ROS-generating agent isadministered prior to photoinactivation of catalase. In other specificembodiments, the ROS-generating agent is administered after thephotoinactivation of catalase. Preferably, the peroxide solution istopically administered (e.g., as a liquid or a spray). Administration ofthe ROS generating agent can be according to all modes of local orsystemic administration known in the art.

In one embodiment, methods of the invention comprising photoinactivationof catalase are directed to an infected external tissue of a subject,including, but not limited to, skin, hair and nails. In otherembodiments, internal tissues, such as gastrointestinal organs orcavities (oral, vaginal or nasal cavities), may be targeted as well.

Peroxide solutions and/or ROS generating agents can be administeredalone or as a component of a pharmaceutical formulation. The compoundsmay be formulated for administration, in any convenient way for use inhuman or veterinary medicine. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, and preservatives can also bepresent in the compositions.

Pharmaceutical formulations of the invention include those suitable forintradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/orintravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, and the particular mode ofadministration, e.g., intradermal.

The formulations can include a pharmaceutically acceptable carrier. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect. Formulations of theinvention can be administered parenterally, intraperitoneally,subcutaneously, topically, orally (e.g., the ROS generating agent) or bylocal administration, such as by aerosol or transdermally. Formulationscan be administered in a variety of unit dosage forms depending upon theseverity of the infection or the site of the infection and the degree ofillness, the general medical condition of each patient, the resultingpreferred method of administration and the like. Details on techniquesfor formulation and administration of pharmaceuticals are well describedin the scientific and patent literature, see, e.g., the latest editionof Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals. Aformulation can be admixtured with nontoxic pharmaceutically acceptableexcipients which are suitable for manufacture. Formulations may compriseone or more diluents, emulsifiers, preservatives, buffers, excipients,etc. and may be provided in such forms as liquids, powders, emulsions,lyophilized powders, sprays, creams, lotions, controlled releaseformulations, tablets, pills, gels, on patches, in implants, etc.

In practicing this invention, the pharmaceutical formulations can bedelivered transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols. In specific embodiments,delivery can be mediated by a transdermal patch, bandage or dressingimpregnated with compositions comprising the peroxide solution and/orROS generating agent. Sustained release can be provided by transdermalpatches, for slow release at the site of infection.

The amount of pharmaceutical formulation adequate to reduce or eradicatepathogenic microbes is a therapeutically effective dose. The dosageschedule and amounts effective for this use, i.e., the dosing regimen,will depend upon a variety of factors, including the stage of theinfection, the severity of the infection, the general state of thepatient's health, the patient's physical status, age and the like. Incalculating the dosage regimen for a patient, the mode of administrationalso is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617:Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra). The state of the art allows theclinician to determine the dosage regimen for each individual patient,active agent and disease or condition treated. Guidelines provided forsimilar compositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods of the invention are correct andappropriate.

Single or multiple administrations of pharmaceutical formulations of theinvention can be given depending on for example: the dosage andfrequency as required and tolerated by the patient, the persistence ofinfection, or lack thereof, after each administration, and the like. Theformulations should provide a sufficient quantity of peroxide solutionto effectively treat, prevent or ameliorate the infection.

Methods of the invention target catalase positive microbes which areassociated with, or may give rise to, infection. Both Gram-negative andGram-positive bacteria serve as infectious pathogens in vertebrateanimals. Such catalase positive Gram-positive bacteria include, but arenot limited to, Staphylococci species. Catalase positive Gram-negativebacteria include, but are not limited to, Escherichia coli, Pasteurellaspecies, Pseudomonas species (e.g., P. aeruginosa), and Salmonellaspecies. Specific examples of infectious catalase positive bacteriainclude but are not limited to, Helicobacter pylori, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria species (e.g. M.tuberculosis complex. M. avium complex, M. gordonae clade, M. kansasiiclade, M. nonchromogenicum/terrae clade, Mycolactone-producingmycobacteria, M. simiac clade, M. abscessus clade, M. chelonae clade, M.fortuitum clade, M. mucogenicum clade, M. parafortuitum clade, M. vaccaeclade, M. ulcerans, M. vanbaalenii, M. gilvum, M. bovis, M. leprae, M.spyrl, M. kms, M. mcs, M. jls, M. intracellulare, and M. gordonae.),Acinetobacter baumannii, Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, pathogenic Campylobacterspecies, Haemophilus influenzae, Bacillus antracis, Corynebacteriumdiphtheriae, corynebacterium species, Erysipelothrix rhusiopathiae,Chlamydia trachomatis, Clostridium perfringers, Clostridium tetani,Klebsiella pneumoniae, Pasteurella multocida, Bacteroides species,Fusobacterium nuclealum, Treponema pallidium, Treponema perlenue,Leplospira, Rickettsia, and Actinomyces israelli, Mycoplasma andChlamydia species.

Examples of catalase positive fungi include, but are not limited to,Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Candida glabrata,Candida tropicalis, Candida parapsilosis, and other catalase-positiveCandida spp. Candida auris, and Trichophyton rubrum.

The light for photoactivation of catalase can be produced and deliveredto the site of infection by any suitable means known in the art.

While it has now been determined that the antimicrobial effect ofphotoinactivation is significantly and unexpectedly increased uponadministration of a low-concentration of H₂O₂ and/or a ROS-generatingagent, the antimicrobial effectiveness is also significantly improvedwhen the light is provided by a pulsed nanosecond laser compared tocontinuous wavelength LED. Accordingly, in specific embodiments, thelight source is a is a pulsed nanosecond laser. Pulsed operation oflasers refers to any laser not classified as continuous wave, so thatthe optical power appears in pulses of some duration at some repetitionrate. Nanosecond laser families can range from the UV to the IR withwavelengths up to 1064 nm, repetition rates up to 2 kHz, and pulseenergy up to 20 mJ. Photoinactivation of catalase can be conducted withlight having a wavelength of about 400 nm to about 460 nm. In specificembodiments, the wavelength is about 400 nm to about 430 nm, applied ata dosage of about 5 J/cm²-to about 200 J/cm².and in other specificembodiments, about 14 J/cm² to about 32 J/cm². In other specificembodiments, the pulse duration is about 5 nanoseconds. Light deliveredin this range by a pulsed nanosecond laser is clinically advantageousbecause thermal damage is minimal, temporary or otherwise non-existent.In more specific embodiments, the wavelength is 410 nm (delivered usingabout 15 J/cm²), applied by a pulsed nanosecond laser according tomethods known in the art for operation of such lasers.

Exposure times range from about 5 to about 10 minutes in length, and canbe repeated weekly as needed, for example, about twice per week forseveral months. In clinical applications, patients may receive treatmentfor between one to 3 months or longer as determined by the practicingphysician.

In other embodiments, photoactivating light can be delivered to the siteof infection through various optical waveguides, such as an opticalfiber or implant. In some embodiments, the photoinactivating light isdelivered by optical fiber devices that directly illuminate the site ofinfection. For example, the light can be delivered by optical fibersthreaded through small gauge hypodermic needles. In addition, light canbe transmitted by percutaneous instrumentation using optical fibers orcannulated waveguides. For open surgical sites, suitable light sourcesinclude broadband conventional light sources, broad arrays of LEDs, anddefocused laser beams. The light source can be operated in theContinuous Wave (CW) mode. Photoinactivation of catalase is preferablyconducted with light having a wavelength of about 400 nm to about 430 nmand a dosage of about 5 J/cm²-to about 200 J/cm².and in specificembodiments, about 14 J/cm² to about 32 J/cm² In other specificembodiments, the wavelength is 410 nm (delivered using about 15 J/cm²),applied by a CW LED according to methods known in the art for operationof such LED sources. Exposure times range from any light source rangefrom about 5 to about 10 minutes in length.

In other embodiments of the invention, the photoinactivation of catalaseis performed on an inanimate surface including but not limited to metal,plastic, fabric, rubber, stone, composite surfaces or wood. In specificembodiments, the inanimate surface comprises objects such asinstruments, catheters, medical and military equipment, furniture,handrails, textiles, fixtures such as sinks and plumbing materials,building materials, industrial or electronic equipment, and food productor food processing equipment. Photoinactivation of catalase on inanimatesurfaces is preferably conducted with light having a wavelength of about400 nm to about 430 nm, and in combination with administration of asolution having a low-concentration of a peroxide. In specificembodiments, the wavelength is 410 nm (delivered at 15 J/cm²), appliedby a pulsed nanosecond laser. Exposure times range from about 5 to about10 minutes in length.

The following examples are put forth for illustrative purposes only andare not intended to limit the scope of what the inventors regard astheir invention.

EXAMPLES

The following materials and methods were employed throughout Examples1.-4.

Bacterial strains: Enterococcus faecalis NR-31970, Enterococcus faecalisHM-325, Escherichia coli BW 25113, Escherichia coli ATCC 25922.Klebsiella pneumoniae ATCC BAA 1706. Klebsiella pneumoniae ATCC BAA1705. Salmonella enterica ATCC 70720. Salmonella enterica ATCC 13076.Acinetobacter baumannii ATCC BAA 1605. Acinetobacter baumannii ATCCBAA-747. Pseudomonas aeruginosa ATCC 47085 (PAO-1). Pseudomonasaeruginosa 1133. Pseudomonas aeruginosa ATCC 15442. Pseudomonasaeruginosa ATCC 9027.

Quantitation of catalase by Amplex red catalase kit: Quantification ofcatalase both from the pure catalase solution and bacteria was achievedby a fluorescent amplex red catalase kit. 25 μl of analyte wereincubated with 25 μl (40 μM of H₂O₂) for 30 min at room temperature.Then 50 μl of working solution (100 μM Amplex Red reagent containing 0.4U/ml horseradish peroxidase) were added to the abovementioned mixture,and the subsequent mixture were incubated for another 30-60 min in thedark. After that, the fluorescence was recorded at an emission of 590 nmwhen excited at 560 nm.

Resonance Raman spectrum of dried catalase film: Catalase was measuredby its Raman peaks at around 1300-1700 cm-1 measured by resonance Ramanspectroscopy (1221, LABRAM HR EVO, Horiba) with a 40× objective(Olympus) and an excitation wavelength of 532 nm. Samples (dried ‘coffeering’ were sandwiched between two glass cover slides (48393-230, VWRinternational) with a spatial distance of ˜80 μm. To study thephotoinactivation (by a continuous-wave LED), the same samples weremeasured after each laser irradiation.

CFU experiments to test the potential synergy between photoinactivationof catalase and H₂O₂: Overnight-cultured bacteria was centrifuged, thesupernatant was discarded, and the pellet was resuspended with the sameamount of PBS. The laser source used in the study is nanosecond (ns)pulsed OPO laser purchased from OPOTEK Inc, model number as OpoletteHE355 LD, having the following key specifications: wavelength range,410-2400 nm; pulse repetition rate, 20 Hz; maximum pulse energy at 460nm, 8 mJ; pulse duration, 5 nanosecond; spectral linewidth, 4-6 cm−1;and pulse-to-pulse stability, <5%. For each bacterial strain there werefour groups: untreated one, ns-410 nm-treated group, H₂O₂ (22 or 44mM)-treated group, ns-410 nm plus H₂O₂ (22 or 44 mM)-treated group. Dosefor ns-410 nm exposure was 15 J/cm². H₂O₂ was incubated with bacteriafor 30 min at 37° C. with the shaking speed of 200 rpm. Afterincubation, bacterial burden from each group was serial diluted,inoculated onto TSA plates, then counted by enumeration of these plates.

CFU experiments to test the potential synergy between photoinactivationof catalase and ROS-generating antibiotics: Overnight-cultured bacteriawas centrifuged, and then the supernatant was discarded and re-suspendedwith the same amount of fresh TSB. Then prior to any treatments, theabove solution was incubated with antibiotics (10 μg/ml) for 1 hour. Foreach bacterial strain, four groups were tested: untreated one, ns-410nm-treated group, antibiotic (2 μg/ml)-treated group, ns-410 nm plusantibiotic (2 μg/ml)-treated group. Dose for ns-410 nm exposure was 15J/cm2. Antibiotic was incubated with bacteria for up to 6 hours at 37°C. with the shaking speed of 200 rpm. At each time interval, bacterialburden from each group was serial diluted, inoculated onto TSA plates,then counted by enumeration of these plates.

Confocal imaging of intracellular bacteria assay: As described elsewhere(Yang, X., et al. International journal of nanomedicine 13, 8095(2018)), murine macrophage cells (RAW 264.7) were cultured in DMEMsupplemented with 10% FBS at 37 degrees C. with CO2 (5%). Cells wereexposed to MRSA USA300 or Salmonella enterica (with/without ns-410 nmexposure) at a multiplicity of infection (MOI) of approximately 100:1 atserum-free DMEM medium. 1 or 2-hour post-infection, RAW 264.7 cells werewashed with gentamicin (50 μg/mL, for one hour) to kill extracellularbacteria in DMEM+10% FBS. After that, RAW 264.7 cells were washed withgentamicin (50 μg/mL) and subsequently lysed using 0.1% Triton-X 100 for3 min. After membrane permeabilization, infected RAW 264.7 cells werestained with Live/Dead stain for 15 min, then samples were fixed in 10%formalin for 10 min prior confocal imaging.

Example 1: Pulsed Blue Laser Effectively Inactivates Pure Catalase andCatalase from Bacteria

Pure catalase solution (bovine liver catalase, 3 mg/ml in the PBS) wasprepared in PBS using a protocol previously published to examine theeffect of 410-nm exposure on the absorption spectrum of catalasesolution (Cheng, L., Photochemistry and Photobiology 34, 125-129(1981)). Catalase shows a pronounced absorption at around 410 nm, andits absorption at this wavelength gradually decreases as the 410-nmexposure elongates (FIG. 1a ). This suggests that the secondarystructure of catalase might be changed, especially in the activeheme-containing domain. In addition, this photoinactivation effect wasexamined by an Amplex Red Catalase kit at different wavelengths (FIG. 1b). The photoinactivation trend is similar to the absorption spectrum ofcatalase, with the 410 nm being the most effective, where 5-min exposuredepleted ˜70% active catalase.

Since most of the aerobic bacteria and facultative anaerobes expresscatalase (Mishra, S. & Imlay, J. Arch Biochem Biophys 525, 145-160,doi:10.1016/j.abb.2012.04.014 (2012)), whether one could photoinactivatecatalase in situ from the catalase-positive bacteria was examined. MRSAUSA300 and P. aeruginosa (PAO-1) were selected as the representative forGram-positive and Gram-negative bacteria, respectively. Noteworthy,catalase from both MRSA USA300 (FIG. 2a ) and P. aeruginosa (FIG. 2b )were photoinactivated by blue light exposure region, especially 410-nmexposure. The dose utilized was about 15 J/cm2, well below the ANSIsafety limit of 200 J/cm², and the specimens were stationary-phasecultured bacteria (˜108 cells/ml). ANSI is the American NationalStandard for Safe Use of Lasers, see ANSI Z136.1, Laser Institute ofAmerica 2014.

To further understand how 410 nm exposure could cause the structuralchange of catalase, Resonant Raman spectroscopy was performed to capturethe Raman signature of dried catalase film (FIG. 3). Apparently, 410 nmexposure significantly drops the Raman intensity at 750 cm⁻¹, and theRaman bands ranging from 1300 cm⁻¹ to 1700 cm⁻¹, which are typicalvibrational bands of heme ring from catalase (Chuang, W.-J., Heldt, J. &Van Wart, H. J. J. o. B. C. Resonance Raman spectra of bovine livercatalase compound II. Similarity of the heme environment to horseradishperoxidase compound II. 264, 14209-14215 (1989)). These data furtherconsolidate the fact that 410 nm exposure could cause structural changeof catalase.

In addition, the efficacy between ns-410 nm and CW-410 nm to inactivatecatalase was compared. ns-410 nm is significantly more effective both inthe pure solution form (FIG. 4a ), or from MRSA USA300 (FIG. 4b ) and P.aeruginosa (FIG. 4c ) compared to CW-410 nm. Moreover, ns-410 exposureeliminates the necessity of heating tissue during future clinical study.

Example 2: Photo-Inactivation of Catalase Sensitizes a Wide Range ofBacteria to Low-Concentration H₂O₂

Catalase is an essential detoxifying enzyme in bacteria encounteringvarious endogenous or exogenous stress (Nakamura, K. et al. Microbiologyand immunology 56, 48-55 (2012)). When the gene encoding the expressionof catalase is mutant, pathogens are more susceptible to theenvironmental stress (Mandell, G. L., J Clin Invest 55, 561-566,doi.10.1172/jci107963 (1975)). Whether exogenous addition oflow-concentration H₂O₂ could eliminate those ‘traumatized’ pathogens wasinvestigated. As shown in FIG. 5, photoinactivation of catalase (15J/cm2) alone didn't reduce the MRSA burden (FIG. 5a ), P. aeruginosaburden (FIG. 5b ), and Salmonella enterica burden (Figure Sc)significantly. Moreover, low-concentration H₂O₂ (22 mM) didn't exert anysignificant antimicrobial effect against both MRSA and P. aeruginosa(FIG. 5). However, subsequent administration of low-concentration H₂O₂after photoinactivation of catalase significantly reduced the MRSA andP. aeruginosa burden (≥3-log 10 reduction, FIG. 5). Interestingly, thebacterial killing trend versus irradiance wavelength is similar to thatof photoinactivation of catalase versus irradiance wavelength.Noteworthy, low-concentration H₂O₂ combined with 410 nm exposure (15J/cm2) achieved total eradication of P. aeruginosa (FIG. 5b ).

Example 3. Photoinactivation of Catalase and Low-concentration HydrogenPeroxide Create a Synergistic Effect

There is a synergistic effect between photoinactivation of catalase andlow-concentration hydrogen peroxide to eliminate stationary-phase MRSAUSA300 and stationary-phase Pseudomonas aeruginosa. FIG. 5a depicts thesynergistic results in a bar-graph. CFU ml−1 (colony-forming unit)designates the bacterial burden. ‘Untreated’ refers to the originalstationary-phase MRSA without any exogenous treatment. ‘H₂O₂ (22 mM,0.075%)’ and ‘ns-light’ refer to stationary-phase MRSA with H₂O₂ and nslight alone, respectively. As shown in the graph, H₂O₂ alone andns-light alone do not exert any significant killing effect on MRSA,however, ns-410 nm in combination with H₂O₂ reduces approximately fourorders of magnitude of bacterial burden. The same phenomenon happenswith other wavelengths as well. Noteworthy, ns-430 nm or ns-430 nmcombined with H₂O₂ reduces around 99% of the bacterial burden under thesame conditions. ns-450 or ns-460 nm combined with H₂O₂ together reducesaround 90% of the bacterial burden. ns-470 nm combined with H₂O₂together reduces around 50% of the bacterial burden. ns-480 nm combinedwith H₂O₂ barely exerts an antimicrobial effect. Altogether, the killingeffect of H₂O₂ is significantly enhanced by blue light photoinactivationof catalase, especially when applied using ns-410 nm. A similarphenomenon occurred with stationary-phase Pseudomonas aeruginosa, whichis a representative of Gram-negative bacteria (FIG. 5b ) and Salmonellaenterica. By employing ns-410 nm combined with H₂O₂ to Salmonellaenterica, an enhanced killing effect of around five orders of magnitudewas observed (FIG. 5c ).

In addition, ns-410 nm combined with H₂O₂ is significantly moreeffective in eliminating microbes compared to CW-410 nm combined withH₂O₂(FIG. 6).

Example 4: Photoinactivation of Catalase Revives ConventionalAntibiotics Against a Wide Range of Bacteria

Besides H₂O₂, whether photoinactivation of catalase could synergize withconventional antibiotics was investigated, especially for antibioticsthat can generate the downstream intracellular ROS. Tobramycin, arepresentative of aminoglycoside, is an example. Tobramycin can inducedownstream ROS burst (Dwyer, D. J. et al. Proceedings of the NationalAcademy of Sciences 111, E2100-E2109, doi:10.1073/pnas.1401876111(2014)), thus the combination of photoinactivation of catalase andtobramycin administration, together, was tested to see whether anenhanced effect was observed.

Interestingly, enhanced killing effect was observed in thecombination-treated group (FIG. 7). More than 100 times enhancementsuggests that photoinactivation of catalase indeed accelerates theantimicrobial effect of ROS-generating antibiotics. As a control, thesame treatment schemes were tested on a catalase-negative Enterococcusstrain, Enterococcus faecalis NR-31970, which did not produce anyenhanced killing effect (FIG. 7). Altogether, this indicates thatphotoinactivation of catalase helps to revive traditional antibioticsagainst catalase-positive pathogens.

Example 5: Photoinactivation of Catalase Assists Macrophage CellsAgainst Intracellular Pathogens

Neutrophils and macrophage cells are highly essential phagocytic cellsthat serve as the first line of defense of the innate immune system(Segal, A. W., Annu Rev Immunol 23, 197-223,doi:10.1146/annurev.immunol.23.021704.115653 (2005)). Catalase, which isencoded by gene, katA, confers indispensable resistance to theantimicrobial agents released by immune cells (Flannagan, R., Heit, B. &Heinrichs, D., Pathogens 4, 826-868 (2015)). Based on these facts, itwas hypothesized that photoinactivation of catalase could assist immunecells to eliminate extracellular and intracellular pathogens. To testthe potential assistance effect, a fluorescent Live/Dead assay was usedto visualize the intracellular live/dead bacteria after ns-410 nmexposure. A higher percent of dead MRSA was observed intracellularly(FIG. 9).

In conclusion, photoinactivation of catalase significantly boosts theefficacy of low-concentration H₂O₂, ROS-generating antibiotics, andimmune cells against broad-spectrum bacteria, including the notoriousdrug-resistant gram-negative bacteria.

The following materials and methods were employed throughout Examples5.-9.

Chemicals and fungal strains: DMSO (W387520, Sigma Aldrich),amphotericin B (A9528-100 MG, Sigma Aldrich), ergosterol (AC1178100050,98%, ACROS Organics). YPD broth (Y1375, Sigma Aldrich). YPD agar (Y1500,Sigma Aldrich). PrestoBlue cell viability assay (A13262, Thermo FisherScientific). Candida albicans SC5314, the test of fungal strains usedsee Table 1.

TABLE 1 Fungal strains utilized for amp B imaging experiments. PathogenStrain Number C. albicans SC5314 SC5314 C. glabrata ATCC2001 ATCC2001 C.tropicalis C22 H3222861 C. parapsilosis C23 F825987 C. lusitaniae C30S1591976 Candida auris Lung From MGH-- commonly used in MKM lab Candidahaemulonii CAU-13 AR-0393 Candida duobushaemulonii CAU-14 AR-0394Candida haemulonii CAU-15 AR-0395 Kodameae ohmeri CAU-16 AR-0396 C.albicans Ca C13 C. albicans Ca C14 C. glabrata Cg C1 C. glabrata Cg C2Candida krusei CAU-17 AR-0397 C. lusitaniae CAU-18 AR-0398 Saccharomycescerevisiae CAU-19 AR-0399 C. albicans Ca C15 C. albicans Ca C16 C.albicans Ca C17 Candida krusei CAU-17 AR-0397 C. lusitaniae CAU-18AR-0398 Saccharomyces cerevisiae CAU-19 AR-0399 C. albicans Ca C15 C.albicans Ca C16 C. albicans Ca C17 Candida auris CAU4 AR-0384 CAU5AR-0385 CAU6 AR-0386 CAU7 AR-0387 CAU8 AR-0388 CAU9 AR-0389

Quantification of catalase from fungus before and after 410 nm exposure:Quantification of catalase both from the pure catalase solution andfungal solution were achieved by a fluorescent amplex red catalase kit.Basically, 25 μl of analyte were incubated with 25 μl (40 μM of H₂O₂)for 30 min at room temperature. Then 50 μl of working solution (100 μMAmplex Red reagent containing 0.4 U/ml horseradish peroxidase) wereadded to the abovementioned mixture, and the subsequent mixture wasincubated for another 30-60 min in the dark. After that, thefluorescence was recorded at an emission of 590 nm when excited at 560nm.

CFU test to quantify the treatment efficacy: Quantification ofantifungal treatment schemes were achieved as following: overnightcultured fungal specimen was washed by sterile PBS. And log-phase fungalpathogens were prepared by dilution into fresh YPD broth at a ratio of1:50 and cultured for another 2-3 hours at 30° C. with the shaking speedof 200 rpm. After that, the fungal concentration was adjusted to bearound 1×108 cells/ml by centrifuging or further dilution with PBS. 10μl of the above fungal solution was exposed to 410 nm for 5 min (150mW/cm2). After that, the exposed sample was collected into 990 μl ofsterile PBS, then supplemented with treatment agents. Later, CFU offungal cells was enumerated by serial dilution and cultured in YPD agarplates for 48 hours.

PrestoBlue viability assay: First log-phase fungal pathogens wereprepared by diluting overnight-cultured fungal pathogens into fresh YPDbroth at a ratio of 1:50 and cultured for another 2-3 hours at 30° C.with the shaking speed of 200 rpm. After that, the fungal concentrationwas adjusted to be around 1×108 cells/ml by centrifuging or furtherdilution with PBS. 10 μl of the above fungal solution was exposed to 410nm for 5 min (150 mW/cm2). After that, the exposed sample was collectedinto 990 μl of sterile PBS, then supplemented with treatment agents.Aliquots were made from the above sample into a 96-well plate, with eachwell containing 100 μl. Then 100 μl sterile YPD broth and 23 μl ofPrestoBlue were added into the same well. Fluorescence signal at 590 nmfrom each well was recorded in a time-course (up to 18 hours with theinterval of 30 min) manner at an excitation of 560 nm. For each strain,in order to know the exact number of fungal pathogens in each well, thecorresponding fluorescence signals were recorded from fungal pathogenswith known numbers, however no external treatments.

Macrophage-Candida albicans interaction unveiled by confocal laserscanning microscopy: As described elsewhere (Yang, X., et al.International journal of nanomedicine 13, 8095 (2018)), murinemacrophage cells (RAW 264.7) were cultured in DMEM supplemented with 10%FBS plus penicillin and streptomycin at 37 C with CO2 (5%). Cells wereexposed to Candida albicans SC5314 (with/without 410 nm exposure) at amultiplicity of infection (MOI) of approximately 10:1 at serum-free DMEMmedium. 1 or 2-hours post-infection, RAW 264.7 cells were washed withgentamicin (50 μg/mL, for one hour) to kill extracellular pathogens inDMEM+10% FBS. After that, RAW 264.7 cells were washed with gentamicin(50 μg/mL) and subsequently lysed using 0.1% Triton-X 100 for 3 min.After membrane permeabilization, infected RAW 264.7 cells were stainedwith Live/Dead stain for 15 min, then samples were fixed in 10% formalinfor 10 min. Formalin was washed away prior confocal imaging.

Example 5: 410 nm Exposure Reduces Intracellular Catalase Amount

It is known that most fungal pathogens are catalase positive (Hansberg,W., et al. Arch Biochem Biophys 525, 170-180 (2012)). To test whether410 nm exposure could cause the loss of catalase activity, the sameapproach to quantify the intracellular catalase amount by the amplex redcatalase kit was utilized before and after 410 nm exposure. Catalasefrom various fungal pathogens, either log-phase or stationery-phasecould be significantly inactivated by 410 nm exposure (FIG. 10).Noteworthy, catalase from notorious Candida auris strain reduced by 60%after only 5-min 410 nm exposure.

Example 7: Photoinactivation of Catalase in Combination with H₂O₂Achieved Total Eradication of C. albicans SC5314 by CFU Assay

Since catalase was effectively inactivated among various fungal strains,whether photoinactivation of catalase could sensitize fungal strains toexternal H₂O₂ attack was investigated. With further administration oflow-concentration H₂O₂ after 410 nm exposure, eradication was achievedafter combinational treatments (FIG. 11). Noteworthy, there was morethan five orders of magnitude enhancement of the function of H₂O₂ afterphotoinactivation of catalase (FIG. 11).

Synergism between photoinactivation of catalase and H₂O₂ to eliminateCandida albicans SC5314 was also observed. The result is shown by ascatter plot in FIG. 12. CFU ml-1 (colony-forming unit) refers to thenumber of bacterial burden. ‘Untreated’ means the originalstationary-phase SC5314 without any exogenous treatment. ‘H₂O₂ (44 mM,0.15%) and ‘ns-light’ means stationary-phase SC5314 with H₂O₂ andns-light alone, respectively. As shown in FIG. 12, H₂O₂ alone andns-light alone doesn't exert significant killing effect on CASC5314,however, ns-light in combination with H₂O₂ reduces around four orders ofmagnitude of bacterial burden. Especially, ns-410 or ns-420, ns-430combined with H₂O₂ achieved total eradication. ns-450 or ns-480 nmcombined with H₂O₂ reduced a similar amount of fungal burden asH₂O₂-alone. Altogether, the killing effect of H₂O₂ is significantlyenhanced by photoinactivation of catalase by blue light, especially byns-410-ns-430 nm. Therefore, an effective synergy exists betweenphotoinactivation of catalase under the blue light range and H₂O₂ toeliminate CASC5314.

Example 8. Photoinactivation of Catalase in Combination with H₂O₂Achieved Efficient Eradication of Broad-spectrum Fungal Species byPrestoBlue Assay

To further confirm that this combinational therapy works as well forother fungal strains, more clinical fungal strains were tested forfeasibility of this synergistic therapy. Unlike bacteria, fungal cellsgrowth is slower, with each colony forming after around 48 hours. Thus,a high-throughput method, PrestoBlue viability assay, was used tomeasure the treatment efficacy. As shown in FIG. 13, the utilization ofPrestoBlue could achieve the same killing effect as the CFU assay.Interestingly, log-phase and stationary-phase CASC5314 demonstratedifferent behavior towards the combinational killing, presumably becauseof the difference in metabolic activity between these two states.However, either log-phase or stationary-phase, photoinactivation ofcatalase always boosts the killing effect of low-concentration H₂O₂.This synergistic therapy was tested among more than twenty differentclinical fungal isolates, and significant killing was consistently foundamong them.

Example 9. Candida auris Strains are Sensitive to 410 nm Light Exposure

Apart from H₂O₂, whether photoinactivation of catalase was capable ofsynergizing with conventional antifungal agents, such as azoles oramphotericin B (amp B) was investigated. Similar to some classes ofantibiotics, amp B kills fungi partly due to the oxidative damage(Belenky, P. et al. Fungicidal drugs induce a common oxidative-damagecellular death pathway. Cell Rep 3, 350-358,doi:10.1016/j.celrep.2012.12.021 (2013). Therefore, to test ourhypothesis, the PrestoBlue assay was conducted after the treatments ofphotoinactivation of catalase and subsequent addition of amp B againstvarious clinical fungal isolates, including C. auris strains.

Interestingly, without the assistance of photoinactivation of catalase,some C. auris strains were resilient to amp B (FIG. 14). Nonetheless,photoinactivation of catalase achieved total eradication of C. aurisstrains regardless of the addition of amp B. Ten C. auris strains weretested and they demonstrated the same behavior. This means C. aurisstrains are exceptionally sensitive to blue light exposure.

Example 10. Photoinactivation of Catalase Inhibits the Formation ofHyphae of C. albicans, and Assists Macrophage Cells to Phagocytose

Host immune cells play important roles against external evasivepathogens. Catalase holds an essential role during the battle between C.albicans and neutrophils or macrophage cells (Pradhan, A. et al.Elevated catalase expression in a fungal pathogen is a double-edgedsword of iron. Plos Pathog 13, e1006405 (2017). Thus, whetherphotoinactivation of catalase could assist macrophage cells against C.albicans was examined. To visualize this effect, RAW 264.7 cells wereinfected with C. albicans and 410 nm-exposed C. albicans at a MOI of 10and labeled with live/dead fluorescence stains.

As shown in FIG. 15, untreated C. albicans stay as hyphae form andpierced through macrophage cells. Whereas 410 nm-exposed C. albicansremained as dead ‘yeast’ form intracellularly.

In summary, photoinactivation of catalase in combination withlow-concentration H₂O₂ presents an effective and novel approach toeliminate broad-spectrum fungus and fungal infections.

Example 11. Photoinactivation of Catalase in Combination With ROSActivating Agent Silver Cation Synergistically Kills Microbes

Electromagnetic energy having a wavelength of ns-410 nm combined with 10μM of silver cation eliminated about 90%/o of MRSA one hour aftertreatment, whereas ns-410 nm alone or silver cation alone does not exertany significant antimicrobial effect (FIG. 16).

Photoinactivation of catalase and low-concentration silver cationsynergistically eliminate E. coli BW25113 as well. The result is shownby scatter plots in FIG. 17. CFU ml−1 (colony-forming unit) isdesignated as the amount of bacterial burden. ‘Untreated’ refers to theoriginal E. coli BW25113 without any exogenous treatment. ‘0.5 μM Ag⁺and ‘CW-410’ or ‘ns-light’ refers to E. coli BW25113 with 0.5 μM Ag⁺ andns-410 alone, respectively. 0.5 μM Ag⁺ alone and CW-410 alone or ns-410alone doesn't exert any significant killing effect on E. coli, however,ns-410 in combination with 0.5 μM Ag⁺ reduces around 99% of bacterialburden (FIG. 17). The same phenomenon happens at other wavelengths aswell. Noteworthy, CW-410 combined with 0.5 μM Ag⁺ didn't significantlyreduce bacterial burden under the same conditions. Our results areconsistent for both 30 and 60 minutes after treatments.

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims. The recitation of a listing of elementsin any definition of a variable herein includes definitions of thatvariable as any single element or combination (or subcombination) oflisted elements. The recitation of an embodiment herein includes thatembodiment as any single embodiment or in combination with any otherembodiments or portions thereof.

REFERENCES

All patents, patent applications and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent and publication was specifically andindividually indicated to be incorporated by reference.

1-19. (canceled)
 20. A method of treating a tissue of a subject infectedwith a catalase-positive microbe, said method comprising the steps of:applying light to the tissue of the subject infected with thecatalase-positive microbe at a wavelength of about 400 nm to about 430nm, wherein the catalase is inactivated, and contacting the tissue witha composition comprising a diluted peroxide solution, thereby treatingthe tissue of the subject infected with the catalase-positive microbe.21. The method of claim 20, wherein the wavelength is about 410 nm. 22.The method of claim 20, wherein the dose of the light is about 5 J/cm²to about 200 J/cm².
 23. The method of claim 20, wherein the dose of thelight is about 15 J/cm².
 24. The method of claim 20, wherein thecatalase-positive microbe is a fungal or bacterial microbe and the lightis provided by a pulsed nanosecond laser.
 25. The method of claim 20,wherein the catalase-positive microbe is a fungal or bacterial microbeand the light is provided by a continuous wave LED.
 26. The method ofclaim 20, wherein the diluted peroxide solution is a hydrogen peroxidesolution.
 27. The method of claim 26, wherein the hydrogen peroxidesolution is between about 0.03% and about 0.3% hydrogen peroxide. 28.The method of claim 27, wherein the hydrogen peroxide solution is 0.3%hydrogen peroxide.
 29. The method of claim 20, further comprisingadministering a reactive oxygen species generating agent to the infectedtissue of the subject.
 30. The method of claim 20, wherein the tissue isskin, scalp or nails.
 31. A method of producing a synergisticantimicrobial effect in a tissue of a subject infected with acatalase-positive microbe, said method comprising the steps of: applyinglight to the tissue of the subject infected with the catalase-positivemicrobe at a wavelength of about 400 nm to about 460 nm, wherein thecatalase is inactivated, and contacting the tissue with a compositioncomprising a diluted peroxide solution, thereby producing thesynergistic antimicrobial effect in the tissue of the subject infectedwith the catalase-positive microbe.
 32. A method of treating a tissue ofa subject infected with a catalase-positive microbe, said methodcomprising the steps of: applying light to the tissue of the subjectinfected with the catalase-positive microbe at a wavelength of about 400nm to about 430 nm, wherein the catalase is inactivated, and contactingthe tissue with a composition comprising a reactive oxygen speciesgenerating agent, thereby treating the tissue of the subject infectedwith the catalase-positive microbe.
 33. The method of claim 32, whereinthe wavelength is about 410 nm.
 34. The method of claim 32, wherein thedose of the light is about 5 J/cm² to about 200 J/cm².
 35. The method ofclaim 32, wherein the dose of the light is about 15 J/cm².
 36. Themethod of claim 32, wherein the catalase-positive microbe is a fungal orbacterial microbe and the light is provided by a pulsed nanosecondlaser.
 37. The method of claim 32, wherein the catalase-positive microbeis a fungal or bacterial microbe and the light is provided by acontinuous wave LED.
 38. The method of claim 32, further comprisingadministering a diluted peroxide solution to the infected tissue of thesubject.
 39. The method of claim 32, wherein the tissue is skin, scalpor nails.